High‐numerical‐aperture (NA; e.g., >1.2 for 60× objective), chromatically corrected (at least PlanApo) objective to capture
as much light as possible, especially important for work that requires subcellular colocalization

Fluorescence microscope: same as in protocol 1, except remove the emission filter from the cube used to excite the donor fluorophore (e.g., for CFP/YFP FRET, remove the
emission filter from the CFP cube); referred to as the FRET cube